Previous studies have demonstrated that the nuclear localization of
ras homolog family member A (RhoA), with prominent concentration in the nucleolus, is a common feature in human
cancer tissues and
cancer cell lines. Although a previous study has demonstrated that the nuclear translocation of RhoA occurs via active transport, a process that occurs through
importin α in a nuclear factor-κB-dependent manner, the mechanism,
biological function and pathological meaning of the nucleolar residency of RhoA remain to be elucidated. As the cell nucleolus is the site of ribosome biosynthesis, the aim of the present study was to investigate the association between
RNA synthesis and the nucleolar localization of RhoA, as well as the molecular mechanisms underlying the residency of RhoA in the nucleolus of HEp-2 (human larynx epithelial
carcinoma) cells. Indirect immunofluorescence microscopy was used to evaluate the subcellular distribution of nuclear RhoA, and immunoblotting analysis was used to determine the total cellular
protein level of RhoA. Consistent with the results of previous studies, untreated HEp-2 cells exhibited bright nucleolar staining, indicating an increased concentration of RhoA in the nucleoli. Treatment with
actinomycin D for the inhibition of
RNA synthesis caused a redistribution of RhoA from the nucleoli to the nucleoplasm with a speckled staining pattern. Immunoblotting revealed that neither the total cellular amount of RhoA nor the integrity of RhoA was affected by treatment with
actinomycin D. In cells that were treated at a decreased concentration (0.05 mg/l) of
actinomycin D, the redistribution of RhoA was reversible following the removal of the
drug from the culture medium. However, this reversal was not observed at an increased
drug concentration (1 mg/l). Overall, to the best of our knowledge, the results of the present study provide the first in situ evidence that the inhibition of
RNA synthesis induces a redistribution of nucleolar RhoA to the nucleoplasm, and additionally suggest that the nucleolar residency of RhoA in HEp-2 cells may be associated with active
RNA synthesis.